The two benchmarking approaches are complementary. Down-sampling uses real somatic mutations but is limited in the parameter regimes it can explore, and it cannot measure specificity directly. In contrast, the virtual tumor approach does not have these limitations. However, it simulates somatic mutations using germline events, which differ from somatic mutations in their nucleotide substitution frequencies and context. As recalibrated base qualities vary for the different bases (owing to biases in machine errors), there is variable sensitivity to detect different substitutions (Supplementary Fig. 2). Because the difference in sensitivity is minimal, we have chosen to use all the germline events. However, it is possible with the virtual tumor approach to simulate the mutation spectrum of a specific tumor type by reweighting the germline events to match the expected mutation spectrum of the tumor.
